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目前,应用于分子水平成像的技术主要有荧光原位杂交(fluorescent in situ hybridization,FISH)、LacO/LacI系统、TetO/TetR系统,以及基于基因编辑技术的锌指蛋白(zinc finger proteins,ZFP)、转录激活因子效应物(transcription activator-like effectors,TALEs)、CRISPR/Cas9系统等。FISH通过结合DNA探针实现对基因结构的成像,但此结合需通过甲醛或加热变性实现,可导致染色体结构的改变,不适用研究活细胞中的天然染色质动力学[14]。LacO/LacI系统和TetO/TetR系统通过在基因位点整合人工DNA序列阵列,使之与带有荧光分子标记的配体结合实现基因组成像,但这些插入的外源序列可能会干扰目标位点并产生干扰[15]。ZFP和TALEs系统都可以标记重复序列,如实现端粒的可视化[16-17],但是,ZFP系统的设计复杂耗时且细胞毒性大,TALEs存在体积大、递送困难的问题。
而CRISPR/Cas9系统可与多种荧光分子融合表达,操作较为简便,定位较为精准,对活细胞生理状态影响较小,在活细胞成像中应用更加灵活,既可有效可视化基因组中的非重复序列,又能实现染色体动力学的实时追踪成像。与其他成像技术相比,基于CRISPR/Cas9的活细胞成像技术具有独特的优势和局限性(见表1)。
表 1 基因组成像方法的优势和局限性
Table 1. Advantages and limitations of genome imaging methods
Imaging techniques Methods Advantages Limitations FISH DNA probe Widely applications; Simple preparation of probe Fixed cells; Potentially DNA damage LacO/LacI system et al. Synthetic DNA sequence Living cell imaging Potentially exogenous sequence interference ZFP Fused with fluorescent protein High specificity; Applied to repeated sequences Complex design; High cytotoxicity; Can’t applied to repeated sequences TALEs Fused with fluorescent protein High specificity; Low cytotoxicity; Applied to repeated sequences Difficulty in delivery; Can’t applied to non-repeated sequences CRISPR/Cas9 system Multiple labeling modes Simple design; Low cytotoxicity; Applicable to various sequences Risk of off-target
Advances in live cell imaging technology of CRISPR/Cas9 system (invited)
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摘要: CRISPR/Cas9系统因其高效、操作简便、物种适应性广等优势被广泛应用于基因编辑领域,该系统是由靶向目标DNA序列的引导RNA (sgRNA)和具有切割酶活性的Cas9蛋白组成。近年来,通过将核酸酶失活的Cas9突变体dCas9 (dead Cas9)或sgRNA与荧光蛋白(FPs)、有机染料、量子点(QDs)结合开发出一系列超分辨活细胞成像技术,该技术有助于在更高分辨率下研究不同基因、染色体以及基因与染色体之间的时空关系,对促进遗传学、细胞生物学和生物医学等领域的快速发展具有重要意义。文中主要总结基于CRISPR/Cas9系统的活细胞成像技术的最新进展,有望进一步扩大活细胞成像技术在生物医学领域的广泛应用。
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关键词:
- 活细胞成像 /
- CRISPR/Cas9 /
- 荧光分子 /
- 超分辨
Abstract: CRISPR/Cas9 system are widely used in gene editing due to its high efficiency, simple operation and wide species adaptability. The system consists of a guide RNA (sgRNA) that targets the target DNA series and Cas9 with cleavage enzyme activity. In recent years, researchers have developed a range of super-resolution live cell imaging techniques by combining nuclease-inactivated Cas9 mutants dCas9 (dead Cas9) or sgRNA with fluorescent proteins (FPs), organic dyes, and quantum dots (QDs). This technology helps researchers to study different genes, chromosomes and the spatio-temporal relationship between genes and chromosomes at higher resolutions, which is of great significance to promote the rapid development of genetics, cell biology and biomedicine. This paper summarizes the advances in live cell imaging technology based on CRISPR/Cas9 system, which is expected to further expand the wide application in the biomedical field.-
Key words:
- live cell imaging /
- CRISPR/Cas9 /
- fluorescent molecules /
- super resolution
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图 1 CRISPR/dCas9介导基于荧光蛋白的成像示意图[19]。(a) dCas9-FP融合蛋白; (b) MCP-MS2-FP系统:融合表达荧光蛋白的MCP适配体与被改造的sgRNA MS2茎环结合; (c) dCas9-GFP和PP7-PCP-RFP系统同时应用:多基因位点多色成像
Figure 1. Schematic diagram of CRISPR/dCas9 mediated fluorescent protein based on imaging[19]. (a) dCas9-FP fusion protein; (b) MCP-MS2-FP system: the MCP aptamer fused with fluorescent protein binds to the modified sgRNA MS2 stem ring; (c) Simultaneous application of dCas9-GFP and PP7-PCP-RFP systems: multicolor imaging of multiple gene loci
图 2 CRISPR/Cas9活细胞荧光成像图[13]。(a) 多个优化的sgRNAs对MUC4内含子的非重复区域进行CRISPR标记成像; (b) RPE细胞端粒的CRISPR成像(比例尺5 μm)和其运动轨迹示踪图(比例尺200 nm);(c) HeLa细胞有丝分裂时MUC4图像
Figure 2. CRISPR/Cas9 fluorescent imaging of living cells[13]. (a) CRISPR labeling of the nonrepetitive region of MUC4 intron using multiple optimized sgRNAs; (b) CRISPR imaging of telomeres in RPE cells (scale bar 5 μm) and its trajectory tracing diagram (scale bar 200 nm); (c) MUC4 image of HeLa cell during mitosis
图 3 CRISPR/dCas9介导基于有机染料的成像示意图[19]。(a) dCas9-HaloTag融合蛋白; (b) sgRNA-Broccoli/DFHBI-1T系统:经修饰的sgRNA用于结合一到多个RNA适配体DFHBI-1T;(c) sg-RNAMTS-MB系统:分子靶标MB与核酸靶点MTS结合激发荧光
Figure 3. Schematic diagram of CRISPR/dCas9 mediated organic dye based imaging[19]. (a) dCas9-HaloTag fusion protein; (b) sgRNA-Bro-ccoli/DFHBI-1T system: modified sgRNA is used to bind one or more RNA aptamers DFHBI-1T; (c) sgRNA-MTS-MB system: molecular biomarker MB combines with nucleic acid target MTS to stimulate fluorescence
图 4 CRISPR/dCas9介导基于量子点(QDs)的成像示意图[19]。 (a) LplA介导量子点系统:通过LplA介导dCas9与TCO2结合并与TZ1-QD反应; (b) BirA介导量子点系统:同过BirA介导dCas9生物素化并与SA-QD结合
Figure 4. Schematic diagram of CRISPR/dCas9 mediated quantum dots (QDs) based imaging[19]. (a) LplA-mediated quantum dots system: dCas9 combines with TCO2 and reacts with TZ1-QD via LplA; (b) BirA-mediated quantum dots system: BirA-mediated dCas9 biotinylated and combines with SA-QD
表 1 基因组成像方法的优势和局限性
Table 1. Advantages and limitations of genome imaging methods
Imaging techniques Methods Advantages Limitations FISH DNA probe Widely applications; Simple preparation of probe Fixed cells; Potentially DNA damage LacO/LacI system et al. Synthetic DNA sequence Living cell imaging Potentially exogenous sequence interference ZFP Fused with fluorescent protein High specificity; Applied to repeated sequences Complex design; High cytotoxicity; Can’t applied to repeated sequences TALEs Fused with fluorescent protein High specificity; Low cytotoxicity; Applied to repeated sequences Difficulty in delivery; Can’t applied to non-repeated sequences CRISPR/Cas9 system Multiple labeling modes Simple design; Low cytotoxicity; Applicable to various sequences Risk of off-target -
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